The present invention relates generally to the field of physiological dysfunctions associated with Pseudoxanthoma elasticum. More particularly, the invention is concerned with the identification of a gene associated with Pseudoxanthoma elasticum, as well as mutations in the gene that cause the disease. The present invention also relates to methods for detecting and diagnosing Pseudoxanthoma elasticum, to methods for identifying carriers of mutant and normal alleles of the gene associated with Pseudoxanthoma elasticum, to methods for screening compounds to identify potential therapeutics for Pseudoxanthoma elasticum, to treatment methods for Pseudoxanthoma elasticum, and to useful cell lines and animal models of the disease.
Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of elastic fibers in skin, arteries and the retina, that result in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency, cardiovascular disease and retinal hemorrhages leading to macular degeneration.
The skin manifestations are among the most common characteristics of PXE, but the ocular and cardiovascular symptoms are responsible for the morbidity of the disease. Characteristic skin lesions are generally an early sign of PXE and were first described by a French dermatologist in 1896. Skin lesions are usually detected during childhood or adolescence and progress slowly and often unpredictably. Therefore, the initial diagnosis of PXE is sometimes made by a dermatologist. The skin lesions consist of yellowish papules and plaques and laxity with loss of elasticity, and result from an accumulation of abnormal mineralized elastic fibers in the mid-dermis. Lesions are typically seen on the face, neck, axilla, antecubital fossa, popliteal fossa, groin and periumbilical areas. A PXE diagnosis can be confirmed by a skin biopsy that shows calcification of fragmented elastic fibers in the mid- and lower dermis.
Another characteristic of PXE is the presence of ocular lesions due to the accumulation of abnormal elastic fibers in the Bruch""s membrane, resulting in angioid streaks. Doyne was the first to describe these ocular streaks in 1889, and Knapp introduced the term xe2x80x9cangioid streaksxe2x80x9d for their resemblance to blood vessels. The combination of PXE and ocular manifestations was initially referred to as the Gronblad-Strandberg syndrome, after the names of two ophthalmologists who independently related the occurrence of angioid streaks to PXE in 1929. The majority of PXE patients (approximately 85%) develop ocular manifestations during their second decade of life. Bilateral angioid streaks are normally seen as linear gray or dark red lines with irregular serrated edges lying beneath normal retinal blood vessels and represent breaks in the Bruch""s membrane. The Bruch""s membrane is not in a true sense a xe2x80x9cmembranexe2x80x9d but rather a heterogeneous elastin-rich layer separating the chorioid from the retina. The elastic laminae of the Bruch""s membrane is located between two layers of collagen (type I, III and IV) which lie in direct contact with the basement membranes of the retinal pigmented epithelium (RPE) and the capillaries in the choriocapillary layer of the chorioid. As a consequence of angioid streaks, a PXE patient progressively develops a chorioidal neovascularization with a subsequent hemorrhagic detachment of the fovea and later scarring. Optic nerve drusen may also be associated with angioid streaks and results in visual field deficits and even advanced visual impairment.
Common cardiovascular complications of PXE are due to the presence of abnormal calcified elastic fibers in the internal elastic lamina of medium-sized arteries. The broad spectrum of phenotypes includes premature atherosclerotic changes, intimal fibroplasia causing angina or intermittent claudication or both, early myocardial infarction and hypertension. Fibrous thickening of the endocardium and atrioventricular valves can also result in restrictive cardiomyopathy. Approximately 10% of PXE patients also develop gastrointestinal bleeding and central nervous system complications (such as stroke and dementia) as a consequence of systemic arterial wall mineralization. In addition, renovascular hypertension and atrial septal aneurysm can be seen in PXE patients.
Strikingly, lung abnormalities are not a significant phenotypic feature of PXE, even though pulmonary tissues are rich in elastic fibers. Mineralization of pulmonary elastic fibers has only been noted in a few patients.
PXE is usually found as a sporadic disorder but examples of both autosomal recessive and autosomal dominant forms of PXE have been reported. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Recent reports have linked both the dominant and recessive forms of PXE to a 5 cM domain on chromosome 16P 13.1 However, the mechanisms underlying the physiological defects characteristic of PXE are not understood.
Therefore, there is a need in the art for methods and compositions for diagnosing and treating PXE and PXE associated phenotypes.
The invention provides methods and compositions for diagnosing and treating PXE and PXE associated physiological dysfunctions. According to the invention, mutations associated with PXE are located in the (MRP6) ABCC6 gene. Therefore, methods for detecting the presence of a mutation associated with PXE involve interrogating the (MRP6) ABCC6 gene, or a portion thereof, for the presence of one or more mutations that are associated with PXE. Accordingly, one aspect of the invention provides methods for identifying individuals that have one or two mutant alleles at the PXE locus. PXE is most often an autosomal recessive disease. Therefore, an individual with two mutant (MRP6) ABCC6 alleles associated with PXE will develop symptoms characteristic of the disease. In contrast, an individual with one mutant (MRP6) ABCC6 allele associated with PXE is a carrier of the disease and does not develop full-blown PXE. However, according to one embodiment of the invention, a PXE carrier may develop mild forms of the characteristic manifestations. Accordingly, a PXE carrier status can be indicative of a predisposition to PXE related symptoms such as eye, skin, or cardiovascular problems. In a preferred embodiment of the invention, genetic counseling is provided to an individual identified as having a mutation associated with PXE in one or both alleles of the PXE ((MRP6) ABCC6) locus.
In another aspect, the invention provides compositions for detecting the presence of a mutation associated with PXE at the (MRP6) ABCC6 locus. In a preferred embodiment, an oligonucleotide that hybridizes to the (MRP6) ABCC6 locus is used in a diagnostic assay. In a more preferred embodiment, the oligonucleotide includes a sequence complementary to a mutation that is associated with PXE. Alternatively, an antibody-based diagnostic assay is used to detect the presence of a mutation associated with PXE at the (MRP6) ABCC6 locus.
Other aspects of the invention include therapeutic uses of the (MRP6) ABCC6 gene or protein, drug screening, the identification of (MRP6) ABCC6 homologues in other organisms (including mammalian organisms), cellular and animal models of PXE, the identification of (MRP6) ABCC6 functional domains related to the PXE phenotype, the identification of regulators of (MRP6) ABCC6 expression (mutations in these regulators can also result in PXE related symptoms), the identification of genes/proteins that interact with (MRP6) ABCC6 (alterations in these interacting molecules can also cause PXE related symptoms).
Thus, in one series of embodiments the invention provides methods for screening for the presence of a PXE mutation by interrogating an MRP6 nucleic acid obtained from a patient for the presence of a PXE mutation. The screen is positive is the presence of a PXE associated mutation is detected. A PXE associated mutation is a mutation that causes the PXE phenotype in an individual that is homozygous for the mutation. PXE associated mutations also causes the PXE phenotype in an individual that is a compound heterozygote with two different mutant alleles at the MRP6 locus, wherein each allele is a PXE associated allele. Nucleic acid is isolated from a patient biological sample, and the biological sample is preferably blood, saliva, amniotic fluid, or tissue such as a biopsy tissue. According to the invention, an MRP6 nucleic acid is a nucleic acid obtained from the MRP6 locus. An MRP6 nucleic acid can be mRNA, genomic DNA or cDNA from the MRP6 locus, or a PCR product of any of the above. According to the invention, the MRP6 locus includes the MRP6 exons, introns, and associated promoter and regulatory sequences in the genome surrounding the MRP6 exons.
In one series of embodiments, a PXE associated mutation is detected in MRP6 using a nucleic acid based detection assay. Preferred nucleic acid based detection assays include hybridization assays, primer extension assays, SSCP, DGGE, RFLP, LCR, DHPLC, and enzymatic cleavage assays. In another series of embodiments, a PXE associated mutation is detected in a protein based detection assay. Preferred protein based detection assays include ELISA and a Western blot assays. In one embodiment of the invention, mutation detection assays are provided to screen the MRP6 locus or a portion thereof to determine whether a mutation is present. The lack of MRP6 expression or the expression of a physically aberrant form of MRP6 may be sufficient to determine that an individual has a PXE associated mutation at the MRP6 locus. Alternatively, the determination that a mutation is present in the MRP6 locus may not be sufficient to determine the PXE status of an individual in the absence of information concerning the specific identity of the mutation. If such a mutation is present, it may be identified according to methods of the invention, for example by sequencing the region of the MRP6 locus that contains the mutation. Once a mutation is identified in a patient sample, the PXE status of the patient can be determined according to methods of the invention. In an alternative embodiment of the invention, specific mutation detection assays are provided to detect a known PXE associated MRP6 mutation in a patient sample.
In another series of embodiments, the invention provides oligonucleotide probes or primers and antibodies for use in mutation detection assays or screens according to the invention.
In another series of embodiments, the invention provides methods for screening candidate drug compounds to identify therapeutic compounds for treating PXE patients (individuals that have PXE due to the presence of two recessive PXE associated MRP6 alleles, or one apparently dominant PXE allele) or PXE carriers (individuals with one normal MRP6 allele and one allele with a PXE associated mutation).
In another series of embodiments, the invention provides methods for treating PXE patients or carriers using a normal MRP6 nucleic acid or protein to restore normal MRP6 function to the individual or to specific cells or tissues or the individual.
In another series of embodiments, the invention provides methods for creating transgenic or knockout cell lines and animals in order to provide a model system for PXE.
In another series of embodiments, the invention provides methods for identifying compounds such as other intracellular proteins that interact with MRP6 thereby to identify additional therapeutic targets for PXE treatment.
Accordingly, the invention provides methods and compositions for unambiguously determining the PXE status of an individual. The invention provides methods for detecting deletions, substitutions, insertions, and rearrangements in the MRP6 locus that are associated with PXE. In preferred embodiments, the invention provides methods for identifying mutations known to be associated with PXE. Preferred mutations include mutations that affect one or more of the bases in codons 1114, 1138, 1141, 1298, 1302, 1303, 1314, 1321 and other codons identified herein as being important for normal MRP6 function. Alternatively, the invention provides methods to identify mutations that result in non-conservative substitutions in the MRP6 locus. In a further embodiment, the invention provides assays to detect PXE associated mutations at intron/exon splice sites of the MRP6 gene. The invention also provides methods to detect mutations that affect one or more regulatory elements of the MRP6 gene, including the promoter, the polyA site and other transcriptional or translational control sequences.
Methods of the invention are also useful to screen a population in order to identify individuals with one or more PXE associated MRP6 alleles. According to the invention, these individuals are provided with appropriate genetic counseling in view of their PXE status.